The Program Project application (Years 06-10) entitled "Signaling of Endothelial Permeability and Lung Vascular Injury" addresses the key mechanisms that mediate the loss of pulmonary vascular barrier function. The Program's overall goal is to define the critical signaling pathways regulating lung vascular permeability via the paracellular and transcellular routes. We have approached this subject in a multi-disciplinary fashion bringing to bear approaches in molecular and cellular biology, biochemistry, and cell imaging and functional analyses of lung vascular barrier function. Project 1 addresses the mechanisms of transport of albumin through pulmonary vascular endothelial cells by a transcellular pathway involving transcytosis under both normal conditions and after sepsis; Project 1 in particular addresses the poorly understood role of caveolae as vesicle carriers in mediating transendothelial transport and the interrelationship between the transcellular and paracellular permeability pathways. Project 2 addresses the posttranslational regulation of iNOS and its rote in the generation of high output nitric oxide, and thereby in increasing endothelial permeability. Project 3 addresses the mechanisms by which thrombin activation of PAR-1 in endothelial cells and its coupling to the heterotrimeric G protein, G13, induces Rho activation, thereby mediating increased lung endothelial permeability. The other important aspect of Project 3 deals with the reversal of the increased permeability response and the signaling mechanisms that are also set into motion, following Gal3 activation, mediating the reannealing of the endothelial barrier. Project 4 addresses the mechanisms of caveolin-l-regulated transcellular transport; i.e., signaling by the heterotrimeric G protein Gi that activates Src kinase and the GTPase dynamin, thereby resulting in the fission of caveolae and engagement of vesicle transport. The Program will be buttressed by the Administrative, Endothelial Cell Culture, Molecular Resources, and Imaging and Physiology Cores. We thus hope to provide new insights into the bases of increased lung vascular permeability to protein and edema formation, the hallmarks of Acute Lung Injury. Moreover, unraveling the signaling pathways that mediate the increased lung vascular permeability will identify rational and novel targets that can then be exploited for therapy.